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| Targets |
PAR2 ( IC50 = 23 nM )
AZ3451 targets protease-activated receptor 2 (PAR2) (binds to a remote allosteric site outside the helical bundle) [1] AZ3451 targets protease-activated receptor 2 (PAR2) (functions as a PAR2 antagonist) [2] |
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| ln Vitro |
AZ3451 is a PAR2 antagonist that affects apoptosis, autophagy, cellular senescence, and the inflammatory response in osteoarthritis (OA). AZ3451 stops cartilage deterioration, IL-1β-induced inflammatory response, and chondrocytes from prematurely senescing. By inducing autophagy, AZ3451 reduces the apoptosis of chondrocytes in vitro. NF-κB, PI3K/AKT/mTOR, and P38/MAPK pathways are all involved in AZ3451's protective effects. [2]
- AZ3451 acts as a PAR2 antagonist, binding to a remote allosteric site outside the helical bundle of PAR2 and preventing structural rearrangements required for receptor activation and signalling [1] - AZ3451 exhibits slow binding kinetics, which is beneficial for competing against the tethered ligand of PAR2 [1] - In IL-1β-stimulated chondrocytes, AZ3451 reduced the expression of inflammatory mediators (iNOS, COX2) and cartilage-degrading enzymes (MMP1, MMP13, ADAMTS5), while increasing the expression of cartilage matrix proteins (Aggrecan, Collagen II) and chondrogenic transcription factor Sox9 [2] - AZ3451 inhibited premature senescence of IL-1β-stimulated chondrocytes, as evidenced by decreased p16INK4a expression and reduced number of SA-β gal-positive cells [2] - AZ3451 activated autophagy in IL-1β-stimulated chondrocytes, increasing the expression of autophagy-related proteins (Atg5, Atg7, Atg12, Beclin1, LC3) and the number of mRFP-GFP-LC3 puncta [2] - AZ3451 attenuated IL-1β-induced chondrocyte apoptosis, characterized by decreased apoptotic rate (Annexin V-FITC/PI staining), reduced TUNEL-positive cells, increased Bcl-2 expression, and decreased BAX, Cyto C, and Cleaved caspase 3 expression; it also restored mitochondrial membrane potential [2] - AZ3451 suppressed the activation of P38/MAPK, NF-κB, and PI3K/AKT/mTOR pathways in IL-1β-stimulated chondrocytes, as indicated by reduced phosphorylation of key pathway proteins and inhibited nuclear translocation of P65 [2] |
| ln Vivo |
In a rat osteoarthritis (OA) model, intra-articular injection of AZ3451 can reduce the cartilage degradation caused by surgery.[/2]
- In a surgery-induced rat osteoarthritis (OA) model, intra-articular injection of AZ3451 ameliorated cartilage degradation, as shown by improved H&E, Toluidine blue, and Safranin O/Fast Green staining results and reduced OARIS scores [2] - AZ3451 decreased the expression of MMP13 and Cleaved-caspase 3, and increased Beclin1 expression in the cartilage tissue of OA rats; it also reduced the number of TUNEL-positive cells in cartilage [2] |
| Enzyme Assay |
- X-ray crystallography assay: PAR2 was crystallized in complex with AZ3451, and the crystal structure was analyzed to determine the binding site. The results showed that AZ3451 binds to a remote allosteric site outside the helical bundle of PAR2, which differs from the orthosteric site of the tethered ligand [1]
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| Cell Assay |
The cartilage tissue of one-week-old Sprague-Dawley rats' knee joints is the source of primary rat chondrocytes. The cartilage pieces undergo sequential digestion using 0.2% trypsin and 0.25% collagenase II dissolved in Dulbecco's Modified Eagle Medium (DMEM) culture medium. The freed chondrocytes are gathered and incubated in DMEM enhanced with 10% FBS, 100 units/ml of penicillin, and 100 μg/ml of streptomycin. The cells are treated with 10 μM AZ3451 in the presence or absence of 10 ng/mL IL-1β up to about 80% confluences. In our experiment, we utilize chondrocytes from passages 1-3 in order to prevent phenotypic loss.
- Chondrocyte culture and treatment: Primary chondrocytes were isolated and stimulated with IL-1β (10 ng/mL) to induce OA-like changes, then treated with AZ3451 at different concentrations for specific durations [2] - Western blot assay: Cell lysates were prepared to detect the expression of inflammatory mediators (iNOS, COX2), cartilage matrix-related proteins (Aggrecan, Collagen II, Sox9), cartilage-degrading enzymes (MMP1, MMP13, ADAMTS5), senescence-related protein (p16INK4a), autophagy-related proteins (Atg5, Atg7, Atg12, Beclin1, LC3), apoptosis-related proteins (Bcl-2, BAX, Cyto C, Cleaved caspase 3), and pathway-related proteins (P38, phospho-P38, PI3K, phospho-PI3K, AKT, phospho-AKT, mTOR, phospho-mTOR, NF-κB P65, phospho-NF-κB P65) [2] - SA-β gal staining assay: Chondrocytes were stained with SA-β gal solution to identify senescent cells, and the number of positive cells was counted [2] - Apoptosis detection assays: Annexin V-FITC/PI double staining was used to detect apoptotic cells via flow cytometry; TUNEL staining was performed to visualize apoptotic cells under fluorescence microscopy [2] - Autophagy detection assay: Chondrocytes were transfected with mRFP-GFP-LC3 adenovirus, and the number of autophagosomes (yellow puncta) and autolysosomes (red puncta) was observed and quantified via fluorescence microscopy [2] - Immunofluorescence assay: Chondrocytes were fixed and stained with anti-P65 antibody to observe the nuclear translocation of P65 under fluorescence microscopy [2] |
| Animal Protocol |
male 8-week-old Sprague–Dawley rats
100 μl (50 μg/ml) intra-articular injection - Rat OA model establishment: Male Sprague-Dawley rats were subjected to surgery to induce OA (anterior cruciate ligament transection and medial meniscectomy). Sham-operated rats served as controls [2] - Drug administration: OA rats were randomly divided into model group and AZ3451 treatment group. AZ3451 was administered via intra-articular injection once a week for 8 weeks; the model group received vehicle injection [2] - Tissue collection and detection: After 8 weeks of treatment, rats were euthanized, and knee joint tissues were collected. Histological staining (H&E, Toluidine blue, Safranin O/Fast Green) was performed to evaluate cartilage morphology; immunohistochemical staining was used to detect the expression of MMP13, Cleaved-caspase 3, and Beclin1 in cartilage; TUNEL staining was performed to detect apoptotic cells in cartilage [2] |
| References | |
| Additional Infomation |
AZ3451 belongs to the benzimidazole class of compounds, with the structure 1H-benzimidazole, substituted at positions 1, 2, and 5 by (1S)-1-cyclohexylethyl, 6-bromo-1,3-benzodioxane-5-yl, and 4-(cyanoanilino)acyl groups, respectively. It is an allosteric regulator of protease-activated receptor 2 (PAR2) (IC50 = 23 nM) and possesses anti-osteoarthritis properties. It also exhibits anti-inflammatory, PAR2 negative allosteric regulation, and autophagy induction effects. It is a secondary amide, nitrile compound, benzimidazole compound, benzodioxane-pentene compound, and organobromine compound.
- AZ3451 is a novel PAR2 allosteric antagonist. PAR2 is a G protein-coupled receptor involved in the development of inflammation and various diseases [1][2] - AZ3451's slow binding kinetics make it an ideal candidate to compete with PAR2 chain ligands [1] - AZ3451 exerts a protective effect on chondrocytes in vitro by inhibiting inflammation, senescence, and apoptosis, and activating autophagy. This process is mediated by the P38/MAPK, NF-κB, and PI3K/AKT/mTOR pathways [2] - AZ3451 has shown potential for treating osteoarthritis and has been demonstrated to improve cartilage destruction in a rat model of osteoarthritis [2] |
| Molecular Formula |
C30H27BRN4O3
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|---|---|
| Molecular Weight |
571.464386224747
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| Exact Mass |
570.13
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| Elemental Analysis |
C, 63.05; H, 4.76; Br, 13.98; N, 9.80; O, 8.40
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| CAS # |
2100284-59-9
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| PubChem CID |
126961335
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| Appearance |
White to light brown solid powder
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| LogP |
6.9
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
38
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| Complexity |
882
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C[C@@H](C1CCCCC1)N2C3=C(C=C(C=C3)C(=O)NC4=CC=C(C=C4)C#N)N=C2C5=CC6=C(C=C5Br)OCO6
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| InChi Key |
FJAOGFGHTPYADT-SFHVURJKSA-N
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| InChi Code |
InChI=1S/C30H27BrN4O3/c1-18(20-5-3-2-4-6-20)35-26-12-9-21(30(36)33-22-10-7-19(16-32)8-11-22)13-25(26)34-29(35)23-14-27-28(15-24(23)31)38-17-37-27/h7-15,18,20H,2-6,17H2,1H3,(H,33,36)/t18-/m0/s1
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| Chemical Name |
2-(6-bromo-1,3-benzodioxol-5-yl)-N-(4-cyanophenyl)-1-[(1S)-1-cyclohexylethyl]benzimidazole-5-carboxamide
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| Synonyms |
AZ-3451; AZ3451; AZ 3451
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ~100 mg/mL (~175.0 mM)
Ethanol: ~100 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (3.64 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7499 mL | 8.7495 mL | 17.4990 mL | |
| 5 mM | 0.3500 mL | 1.7499 mL | 3.4998 mL | |
| 10 mM | 0.1750 mL | 0.8750 mL | 1.7499 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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